engleski

Development of novel surface display expression systems in Pichia pastoris

Pichia pastoris (Komagataella sp.) is widely used for high level production of heterologous proteins for more than two decades. More than 600 of heterologous proteins have been expressed in this methylotropic yeast. Here we present development of novel surface display expression system in P. pastoris. For the immobilization of protein of interest on the surface of P. pastoris, S. cerevisiae cell wall proteins Pir4 and Ccw12 were used. These two proteins were previously used for successful surface display of heterologous proteins in S. cerevisiae. To investigate whether these two surface display systems could be used in P. pastoris, two novel genetic cassettes containing host promoter PAOX1, a signal sequence for directing the protein into the secretory pathway, an anchoring domain of S. cerevisiae cell wall proteins for C- or N- terminal immobilization, and genetic tags for detection of the recombinant protein were constructed. Plasmid pBSY3ZPir4 was constructed for N-terminal immobilization of protein and it contains PIR4 gene under a PAOX1 promoter followed by the spacer region (a stretch of eight serine residues), a region consisting of several restriction sites for the insertion of the gene of interest and followed by the -6xHis and -HA tags. For C-terminal immobilization, plasmid pBSY3ZCcw12 containing PAOX1 promoter followed by the part of CCW12 which is coding for the signal sequence, the -HA tag, restriction sites for the insertion of the gene of interest, the part of the CCW12 which is coding for the GPI anchoring signal, and the downstream genetic elements of the CCW12 was constructed. S. cerevisiae gene GRE3 coding for intracellular xylose reductase (XR) was used as a reporter gene. Surface display of XR in P. pastoris, using either of developed systems, was confirmed by western blot and by measurement of the recombinant XR enzyme activity.

Surface display ; Pir4 ; Ccw12 ; Xylose reductase ; Pichia pastoris

nije evidentirano